Heparin, a sulfated glycosaminoglycan produced by mammalian mast cells, has been used extensively during the last 50 years as an anticoagulant for the prevention of post-operative thrombosis and the treatment of acute venous thrombosis. Heparin predominantly exerts its anticoagulant activity by accelerating the ability of the protease inhibitor antithrombin III to inactivate a number of key proteases in the coagulation cascade (1, 2), most notably factor Xa and thrombin, thrombin being the terminal enzyme in the cascade which converts soluble fibrinogen to insoluble fibrin. In fact, a specific pentasaccharide has been identified in heparin which binds with high affinity to antithrombin III and produces a conformational change in the inhibitor (3). This pentasaccharide is sufficient to enhance inactivation of proteases, such as factor Xa, by antithrombin III. In contrast, heparin needs to crosslink antithrombin III with thrombin for enhanced thrombin inactivation to occur (3).
Despite its widespread clinical use as an anticoagulant, however, heparin suffers from a number of major disadvantages. First, it is structurally extremely diverse, varying in its molecular weight, monosaccharide sequence and sulfation pattern (4). As a result of this diversity, less than 50% of heparin molecules in most preparations actually contain the antithrombin III binding pentasaccharide (1-3). Thus, the quality control of different heparin batches is difficult. Second, heparin is an animal byproduct and consequently suffers from the risk of contamination with animal pathogens, a major concern for present day regulatory agencies. Third, heparin can only be administered intravenously as, due to its high molecular weight, it exhibits poor bioavailability when injected subcutaneously (5, 6). Also, the high molecular weight of heparin (10-15kDa) precludes effective oral delivery of the drug. Fourth, in terms of clinical efficacy, heparin exhibits an extremely steep dose response curve. Thus, the coagulation time of patient's plasma must be continually monitored to ensure that drug overdose does not occur. Administration of appropriate heparin doses is further confounded by significant patient variability in heparin responsiveness. These difficulties lead to unacceptable bleeding being a complication of heparin therapy, particularly when the drug is being used as a long term treatment (5, 6). Finally, a significant number of patients develop heparin-induced thrombocytopenia (HIT) following prolonged heparin exposure (5, 6), a condition which precludes future heparin use in these individuals.
In an attempt to overcome some of the problems associated with heparin, a number of low molecular weight (LMW) (mol. wt. 4000-6500) heparin preparations have been developed and licenced (5, 6). These preparations have retained the anti-factor Xa and anti-thrombotic activities of native heparin but are less potent anticoagulants. As a result, the LMW heparins are less likely to induce bleeding complications in patients. Furthermore, due to their smaller size, they have improved bioavailability and can be administered subcutaneously.
An alternative approach has been to use the related glycosaminoglycan, dermatan sulfate, as an antithrombotic agent (7). Interestingly dernatan sulfate, unlike heparin, catalyses thrombin inhibition by a second natural inhibitor of thrombin, heparin cofactor II (8). However, despite the availability of LMW heparins and dermatan sulfate, there is still a considerable need for structurally well-defined anticoagulants and/or antithrombotics which are not animal derived, give reproducible patient responses, can be orally administered and are not prone to inducing thrombocytopenia.
Prior International Patent Application No. PCT/AU96/00238 discloses the preparation of a class of sulfated oligosaccharides based on polymers of monosaccharide units linked by 1.fwdarw.2, 1.fwdarw.3, 1.fwdarw.4 and/or 1.fwdarw.6 glycosidic bonds and consisting of from 3 to 8 monosaccharide units, and the use of these sulfated oligosaccharides as potent inhibitors of human angiogenesis, tumour metastasis and inflammation.
In work leading to the present invention, it has been shown that these sulfated oligosaccharides may be used as anticoagulant/antithrombotic agents to overcome the many problems associated with glycosaminoglycan-derived anticoagulants/antithrombotics. In particular, it has been shown that the sulfated penta- or hexa-saccharides possess a well defined structure, a broad therapeutic window, a highly reproducible patient response, a probably reduced chance of inducing HIT-like syndromes and the potential for oral delivery. Furthermore, these sulfated oligosaccharides do not act via antithrombin III but appear to inhibit coagulation by activating heparin cofactor II. Thus, the active sulfated oligosaccharides to some extent resemble the sulfated polysaccharide dermatan sulfate, rather than heparin, in their mode of action. Such oligosaccharides may be used for both prophylaxis and treatment of many thrombotic and cardiovascular diseases, the most notable of these being deep venous thrombosis, pulmonary embolism, thrombotic stroke, peripheral arterial thrombosis, unstable angina and myocardial infarction. Furthermore, effective oral delivery of the sulfated oligosaccharides makes these agents an alternative to warfarin, a widely used oral anticoagulant with severe side effects.